Physical and functional interactions between gene products can be uncovered through classical genetic screens and through high throughput genetic screens involving mutations or deletions of one or more genes. In standard genetic screen experiments, a gene is mutated or deleted and the resulting phenotype is assessed. High throughput genetic screens rely on the screening of hundreds, thousands or more mutants simultaneously for phenotypes of interest.
Libraries of strains, each deleted for any one of several thousand genes, are known in the art. For example, the Saccharomyces Genome Project has revealed the presence of more than 6000 open reading frames (ORFs) in the S. cerevisiae genome. Many of these genes have been disrupted and replaced with the KanMX (KAN) cassette, which confers resistance to the antibiotic G418 (GENETICIN®). (See Saccharomyces Genome Deletion Project, Stanford University, sequence.stanford.edu/group/yeast_deletion_project.deletion3.html). Libraries can be made using any suitable markers for selection known in the art. For example, the markers may confer resistance to the antibiotics hygromycin B (HygR), nourseothricin (NatR) and bialaphos (PAT). Often the markers NatMX (NAT) and KanMX (KAN) are used. NAT and KAN-marked deletion strains can be obtained by any suitable method known in the art. For example, the strains may be pre-existing or may be constructed de novo a variety of methods. For an example of single- and double-mutant strain construction see Daniel J A, Yoo J, Bettinger B T, Amberg D C, Burke D J. Eliminating gene conversion improves high-throughput genetics in Saccharomyces cerevisiae. Genetics, 2006 January, 172(1):709-711.
The yeast knockout (YKO) library contains specific “molecular barcodes” referred to as “uptags” and “downtags.” These are short (typically 20 base pair) DNA sequences placed within the genome of each deletion strain adjacent to the deletion locus. Each barcode sequence is associated with only one deletion locus, and the sequence can be used to reveal which gene has been deleted within a particular cell. Uptags are flanked by the same pair of sequences, permitting the multiplex amplification of all uptags using a single PCR primer pair. Similarly, downtags are flanked by the same pair of sequences (which differ from the pair of sequences flanking the uptag barcodes), permitting the multiplex amplification of all downtags using a single PCR primer pair. Microarray technology has been used in conjunction with these barcodes to assist in the identification of large numbers of mutants and their respective phenotypes (e.g., relative growth rate under a particular growth condition) (For example, see Giaever G et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature, 2002 Jul. 25, 418(6896):387-391). In this approach, a population of barcoded deletion strains are mixed and grown under selective conditions; a multiplex amplification of barcodes is performed for cells both pre- and post-selection; amplified barcodes from pre- and post-selection are obtained and distinguishably labeled; a microarray displaying sequences complementary to barcode sequences is then used to quantify the relative abundance of barcodes and thus the effect of selection on the relative abundance of each strain type. Other methods known in the art can be used to produce arbitrarily large collections of yeast strains that each carry a unique DNA barcode sequence. A given genomic alteration carried by such a bar-coded strain may thus be uniquely identified. See Yan et al. Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers. Nat. Meth. (2008) 5(8):719-725.
One high-throughput method of uncovering genetic interactions is the synthetic genetic array (SGA) analysis method. This selection involves the mating of one mutant strain carrying a particular marker to an entire library of yeast deletion strains carrying a second marker to generate diploid strains which are heterozygous at two different loci for two different mutations of interest. Heterozygous diploid strains can then be sporulated, and haploid double mutants are specifically recovered after sporulation by virtue of independently selectable markers linked to each of the mutations of interest, by virtue of a selectable marker gene that is specifically expressed in haploids of a particular mating type, and by virtue of one or more negatively selectable markers that are necessarily present in all diploid cells but not in all haploid cells. The effect of having both mutations on yeast cell survival is then determined by measuring the size of the yeast colonies grown on a plate. The growth of these strains provides an indication of the degree of interaction of the combined effect of the two mutant strains on yeast cell growth. Growth of the colonies is quantified by photographing the plates containing the yeast and measuring the size of the colonies. See Tong et al. Global mapping of the yeast genetic interaction network. Science, 2004, 303:808:813.
A related high throughput genetic screen used to probe genome-wide genetic interactions is called dSLAM, (diploid-based synthetic lethality analysis on microarrays) (See Pan X, Yuan D S, Ooi S L, Wang X, Sookhai-Mahadeo S, Meluh P, Boeke J D. dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae. Methods, 2007 February, 41(2):206-221; Pan X, Ye P, Yuan D S, Wang X, Bader J S, Boeke J D. A DNA integrity network in the yeast Saccharomyces cerevisiae. Cell, 2006 Mar. 10, 124(5):1069-1081; Pan X, Yuan D S, Xiang D, Wang X, Sookhai-Mahadeo S, Bader J S, Hieter P, Spencer F, Boeke J D. A robust toolkit for functional profiling of the yeast genome. Mol Cell. 2004 Nov. 5, 16(3):487-496.). In dSLAM, the relative growth rate of mutant yeast strains grown in competition is measured using molecular barcodes and microarray detection. The process relies on creating a population of double mutant strains via en masse transformation of a knockout cassette targeting a particular gene of interest into a library of heterozygote diploid yeast knockout strains. Each double mutant contains the mutation of a particular gene of interest (the ‘query allele’) in combination with a mutant from the library (‘the array allele’). Once this strain pool is created, it is then sporulated and grown on selective media to obtain the corresponding population of haploid double-mutant cells (haploid selection is essentially as described above for SGA). The population of haploid double-mutant cells is then placed under selective growth conditions to determine if the combination mutant has selective growth advantages or disadvantages compared to the single mutant or wild-type. After sporulation and selection, genomic DNA is prepared and the molecular barcodes are PCR amplified in the presence of labeled primers. The DNA derived from the double mutants can be amplified in the presence of Cy3 and the DNA from the control (single mutants) can be amplified in the presence of Cy5. Subsequent analysis using microarrays displaying oligos complementary to barcodes is used to decipher the relative abundance of yeast cells containing double mutants versus the abundance of yeast cells containing single mutants. Unfortunately, the dSLAM method is limited in that every strain in the population under study must share a particular mutation or ‘query allele’ of interest.
Despite the large scale successes of recent breakthroughs in high-throughput screening and selection, there remains a need in the art for techniques which allow screening of even larger numbers of combination mutants and faster and less expensive methods to perform such screens. It would be advantageous to have a method of high-throughput screening that can be used in combination with DNA barcoding technology in which multiple barcode sequences contained at non-adjacent or unlinked loci within a single cell can be fused to generate ‘stitched barcodes’ such that each stitched barcode uniquely identifies a particular combination of distinct genetic alterations.